Ct, IL-18 polymorphism, and laboratory biomarkers for predicting chemosensory dysfunctions and mortality in COVID-19

Aim Patients with COVID-19 often experience chemosensory dysfunction. This research intends to uncover the association of RT-PCR Ct value with chemosensory dysfunctions and SpO2. This study also aims to investigate Ct, SpO2, CRP, D-dimer, and -607 IL-18 T/G polymorphism in order to find out predictors of chemosensory dysfunctions and mortality. Materials & methods This study included 120 COVID-19 patients, of which 54 were mild, 40 were severe and 26 were critical. CRP, D-dimer, RT-PCR, and IL-18 polymorphism were evaluated. Results & conclusion: Low Ct was associated with SpO2 dropping and chemosensory dysfunctions. IL-18 T/G polymorphism did not show an association with COVID-19 mortality; conversely, age, BMI, D-dimer and Ct values did.

Comparison among groups was done via one-way ANOVA. Tukey test was applied as a post-hoc test for multiple comparisons. Data were presented as mean ± standard error of the mean (SEM), Probability (p)-value less than 0.05 was considered significant. CRP: C-reactive protein; Ct: Cyclic threshold; F: Female; M: Male; SpO2: Oxygen saturation level.
to predict COVID-19 mortality. According to our knowledge, no article has examined the association between the -607 T/G SNP of the IL-18 gene and mortality in COVID-19. Therefore, this study was conducted to assess the association between the -607 T/G polymorphism of the IL-18 gene and the mortality risk in the Kurdish population.

Patients
From 1 May to 30 May 2021, 120 COVID-19 patients were recruited, with 60 (50%) males and 60 (50%) females, as displayed in Table 1. Participants of this study originated from several cities in Northern Iraq and displayed signs and symptoms corresponding to SARS-CoV-2 infection. The infection was confirmed via RT-PCR. As per Chinese guidelines, the COVID-19 participants were categorized into three groups: "mild" 54 (45%), "severe" 40 (33.33%), and "critical" 26 (21.67%) [32,33]. The protocol that divided COVID-19 patients into mild, moderate, severe, and critical groups was slightly modified for the current study; the mild and moderate groups were merged into the mild group. According to this protocol, mild cases either don't exhibit pneumonia or exhibit mild pneumonia; severe cases manifest pneumonia characterized by shortness of breath and their SpO 2 <93 and critical patients suffer from respiratory failure, septic shock, or multiple organ failure [33]. During the thirty-day follow-up, all patients in the critical group passed away in the hospital. All COVID-19 patients were non-vaccinated; the patients did not involve in any therapeutic intervention. For the purpose of genotyping, the COVID-19 study participants were re-categorized into two groups based on mortality: those who survived (mild and severe) (n = 94) and those who did not survive (critical) (n = 26). Patients who did not have access to clinical information or did not have a blood or swab sample were excluded.

Sample & data collection
Blood samples were taken from all the participants prior to the administration of any drugs. An oro-nasopharyngeal swab (eSwab, Copan, USA) was taken from the back of the throat and the nasopharynx to investigate the SARS-CoV-2 virus [34]. The emergency departments of the hospital provided data concerning COVID-19, such as comorbidities, demographics, and medical history. In terms of comorbidities, COVID-19 patients were found to have hypertension (HTN), diabetes mellitus (DM), rheumatoid arthritis (RA), and cancer. 78 patients (65%) didn't have any other underlying disease. While 42 patients (35%) had a personal history of comorbidity. HTN was found to be the most regularly occurring comorbidity among COVID-19 cases, with 42.85% (n = 18). DM, on the other hand, was seen in 19.08% of cases (n = 8). Ten people with COVID-19 (23.81%) reported having more than two comorbidities. Cancer was seen in two cases, with a rate of 4.76 percent, while other diseases occurred in four cases at a rate of 9.53 percent.
Methods for measurement of parameters D-dimer and CRP were evaluated by Cobas c311 (Roche, Germany). A Masimo pulse oximeter was utilized to measure SpO 2 . The SARS-CoV-2 detection was conducted via RT-PCR (Bio-Rad, USA); all the procedures from RNA extraction, cDNA synthesis, and virus detection in the swab were done by Bio-Rad reliance SARS-CoV-2 RT-PCR assay kit (Bio-Rad, USA).

DNA extraction & genotyping of IL-18 rs1946518 SNP
As instructed by the manufacturer, a DNA extract kit (Qiagen, Germany) was used to extract DNA from whole blood. The primers used in this study were designed on this website: http://primer1.soton.ac.uk/primer1.html.
The PCR products were then visualized on a 2% agarose gel electrophoresis stained with ethidium bromide and examined under ultraviolet light (Brown, 2016). Gel electrophoresis results of the TG genotype revealed three bands (440 bp, 208 bp, 278 bp), the TT genotype yielded two bands (440 bp and 208 bp) and the GG genotype displayed two bands (440 bp and 278 bp) (Supplementary Figure 1). For ensuring of the result, 20% of samples were sent for sanger sequencing (Macrogen, Korea), only outer forward and reverse primers were used for sequencing. The result of the sanger sequence was shown on the NCBI via the accession code (OP896193 and OP896194) (not released yet). It was analyzed via the Geneious prime program and confirmed by NCBI nucleotide blast (Supplementary Figure 2). NC 000011.10 was used as a reference sequence (https://www.ncbi.nlm.nih.gov/nucleotide/NC 00001 1.10?report=genbank&log$=nuclalign&blast rank=1&RID=SCAV7SFP01N).

Statistical analysis
The SPSS 28 (IBM, USA), GraphPad Prism 9 (GraphPad Software, Inc., USA), and MedCalc 20 (MedCalc Software, Ltd., Belgium) were used for statistical analysis and making graphs. All of the data was found to be in accordance with the Shapiro-Wilk and D'Agostino normality tests, as well as the Levene's test which showed homogeneity of variance. A two-group comparison was conducted using an independent t-test, while a one-way ANOVA was used for comparing more than two groups. A Tukey test was implemented as a post-hoc test for multiple comparisons.
Pearson correlation analysis was used to determine linear relationships between quantitative variables. Binary univariate logistic and multiple regression were used for the prediction of variables in severity and mortality. The receiver operating characteristic (ROC) curve was utilized in order to predict mortality. The mortality prognosis of laboratory parameters was established by means of the cut-off (with the use of Youden index) from the ROC curve. The predictive accuracy of biomarkers was assessed through positive predictive value (PPV) and negative predictive value (NPV). The Chi-square (χ 2 ) test was applied to assess the COVID-19 genotype and allele frequencies. A p-value of 0.05 or below was considered being statistically significant.

Comparison of demographics & laboratory markers
Age and CRP were much higher in the severe and critical groups of COVID-19 than in the mild group (Table 1, Figure 1a and e). Concerning BMI, our results showed that statistical significance (p = 0.02) was only found between the severe and mild groups (Table 1, Figure 1b). In the mild, severe, and critical groups, the average amount of D-dimer was (0.450 ± 0.100), (10.12 ± 3.284), and (46.60 ± 7.883), respectively. The difference in D-dimer between the mild and severe groups was not statistically significant, but there were significant differences between the other categories (Table 1, Figure 1c). Compared with the mild group, severe and critical COVID-19 showed a significant (p < 0.0001) drop in SpO 2 (Table 1, Figure 1d).

Comparison of Ct value among groups of COVID-19 patients
Among COVID-19 patients, the Ct value decreased significantly in the critical groups compared with the mild and severe groups (p = 0.025 and p < 0.0001, respectively). In contrast, there were no statistically significant differences in the Ct value between the mild and severe groups (p = 0.541) (Table 1, Figure 1). Ct values for male patients (22.22 ± 1.113) and female patients (22.24 ± 0.961) were not significantly different (p = 0.992) (Table 2, Figure 2a). Patients who survived had a Ct value of 24.05 0.455, while those who did not had a Ct value of 15.64 ± 2.139. There was a statistically significant difference (p < 0.0001) between them (Table 2, Figure 2b).   Comorbid patients had a mean Ct value of 20.54 ± 1.461, while non-comorbid patients had a mean Ct value of 23.14 ± 0.776; there was no significant difference between them (p = 0.089) (    Figure 2e).

Binary univariate regression model for GD & OD
Ct value was incorporated into a binary univariate logistic regression model to predict OD and GD. RT-PCR Ct values are independent predictors of OD and GD. As shown in Table 3

Multiple linear regression model for SpO 2
To find out if there is an association between lab parameters and the risk of SpO 2 dropping. The models were made with multiple linear regression, and the "stepwise" method was used to choose the variables that went into the models and acted as predictors. The calibration of the model was checked by determining whether the related variables had multicollinearity (tolerance and variance inflation factor (VIF)). By doing stepwise multiple linear regression analysis on age, BMI, D-dimer, CRP, Ct value, and SpO 2 , we found that model 1 (Ct value alone), model 2 (Ct value and age), model 3 (Ct value, age, and BMI), and model 4 (Ct value, age, BMI, and D-dimer) were strongly associated to SpO 2 dropping in COVID-19 patients. Simultaneously, the inclusion of CRP did not enhance the regression model, and as a result, it was omitted from the stepwise analysis. As shown in Table 4, age, BMI, and D-dimer were predictors for SpO 2 dropping. While the Ct value could predict the rise in SpO 2 .

ROC curve analysis as a predictor of mortality in COVID-19
In Table 6 and Figure 4, ROC curves were constructed for the predicting mortality of 120 COVID-19 patients.
future science group 10.2144/fsoa-2022-0082   The results also showed that the AUC with the best performance in biomarkers was the combination of Ct value, D-dimer, CRP, and SpO 2 . They allow us to predict the mortality of the disease with a sensitivity of 92.31% and a specificity of 100%; their NPV and PPV were 97.9 and 100%, respectively. A Ct value of 20.47 is the cutoff point for hospital mortality prediction. The ideal cutoff point for predicting mortality for D-dimer, CRP, and SpO 2 was 6.81μg/ml, 33.56 mg/l, and 70%, respectively.  there was no statistical difference in the frequencies of the two alleles (T vs G: OR = 0.866, 95% CI: 0.476-1.635; p = 0.749) ( Table 7).

Discussion
The respiratory and cardiovascular systems are the primary target of SARS-CoV-2, yet, there is a significant amount of proof to support the importance of chemosensory dysfunctions when assessing and diagnosing COVID-19 patients. The decreased sense of taste and smell in the current COVID-19 pandemic strongly shows a SARS-CoV-2 infection. Furthermore, in addition to standard laboratory values (D-dimer and CRP), parameters such as SpO 2 and the Ct value of the RT-PCR should be considered [35][36][37][38].   Chi-square ( 2 ) was used for associating genotypes and alleles between groups. A probability (p)-value of less than 0.05 was considered significant. CI: Confidence interval; OR: Odds ratio.
Our results revealed the patients who exhibited severe and critical symptoms had a higher CRP than mild cases of COVID-19, CRP levels were associated negatively with SpO 2 ; a published paper confirms that this result reported that an exaggerated elevation of CRP in patients with COPD was negatively correlated with SpO 2 [39]. Despite elevation of CRP in critical COVID-19 patients, but our regression model didn't support its use as predictor for mortality, this finding is in contrast to Huyut MT, Ilkbahar F [40] and Xie Jet al. [41] who displayed that CRP was an important predictor for the progression of COVID-19 disease.
D-dimer levels increased in COVID-19's severe and critical groups. This result parallels a published paper by Yu H-Het al. [42], who documented that D-dimer was higher in patients suffering from severe COVID-19 [42]. D-dimer levels correlated negatively with SpO 2 ; therefore, D-dimer may be a precise target to be assessed to indicate the mortality, as seen in the ROC curve analysis of this study. Yalçin, 2020 also confirmed our result; D-dimer and SpO 2 were shown to be negatively correlated by him [43]. Huyut MTet al. [44] also documented that D-dimer was a predictor for mortality in COVID-19. 10.2144/fsoa-2022-0082 Future Sci. OA (2023) FSO838 future science group A low Ct value was found in COVID-19 patients suffering from GD and GD. This result is parallel with many papers that were done in this field [45][46][47]. There are many explanations for virus-induced GD and OD. First, the virus induces stomatitis and rhinitis, that leads to the breakdown of the neurosensory cells by the antibody. Second, the virus attacks the central nervous system, peripheral nervous system, cerebral cortex, and exclusively cranial nerves related to taste and smell. Lastly, the virus attacks directly ACE 2 expressed on olfactory epithelium and taste buds [48]. The Ct value did not show the gender-based difference in the current study. This result coincides with another finding [49]. However, in COVID-19, The males have been infected more severely than females [50]; the increased mortality in a male might not be because of the viral load, but it may be other reasons: first, the bad habits of males such as smoking and drinking [51]; second, the testosterone in the male has immunosuppressive activity and increases the expression of TMPRSS 2 which is the co-receptor for binding SRAS-CoV-2 virus. In addition, the estrogen in the female has immune-boosting properties against the virus [52]. There was a variation in the immune response dependent on gender. Suppressing toll-like receptor (TLR)-induced interferon (IFN) release decreases the male's capability to eradicate the virus [53,54].
The Ct value was lower in non-survivor patients in this study since the high load of the virus may lead to damage to the lung and induce pneumonia [55]. The multiple regression and correlation in this study showed that the Ct value associated with the dropping level of SpO 2 . There are many challenges in adding CT values to clinical reports: there is no industry-wide standard cut-off; it may vary depending on the type of commercial kits and the stage of the disease; and finally, the ability of the healthcare provider taking the swab and the COVID-19 patients' tolerance may also affect CT values [56]. After addressing these issues and enhancing RT-PCR' sensitivity, adding the Ct value to the results of the test might be an excellent decision.
SpO 2 assesses the respiratory function and arterial oxygenation of COVID-19 and its level decreased in severe and critical groups compared with mild groups. Correlation showed that age, BMI, CRP, and D-dimer were negatively associated, while Ct value was positively associated with a level of SpO 2 . The reason for the association of age with COVID-19 mortality could be due to the presence of comorbidities in older age [57]. An age related malfunction in T-lymphocyte and B-lymphocyte cells results in their inability to clear the virus [58]. Obesity is one of the risk factors for increased mortality in COVID-19 [59,60]. When COVID-19 patients have a high BMI, pro-inflammatory cytokines are also increased, causing damage to lung cells and a decline in SpO 2 levels [61][62][63].
This study evaluated the association of polymorphisms of -607 SNP of IL-18 with COVID-19 mortality among the Kurdish population. The -607 SNP of IL-18 changes the immune response to many viruses, including hepatitis B virus [64], hepatitis C virus, cytomegalovirus [65], and human immune deficiency virus [66]. The cytokine genes are very polymorphic that may change the concentration of cytokine; IL-18 has -607 SNP in the promoter that causes elevation of IL-18. Serum elevation of IL-18 can be regarded as a bad prognostic factor in COVID-19 in the Brazilian population [67]. The current study documented that -607 SNP of IL-18 was not linked to mortality in COVID-19. Chen W-Jet al. [68] proved that the TG genotype of -607 SNP of IL-18 was associated with viral shedding in SARS-CoV-1.
This study has some limitations. First, the sample size is relatively small, if the sample was taken from all governments in Iraq so that the sample represents the population of Iraq. Second, the blood was only taken at the time of admission; it was better to retake blood at regular intervals. Third, healthy controls were not enrolled in the current study. Finally, reports of the prevalence of olfactory and taste disturbances in patients with SARS-CoV-2 have been based mainly on questionnaires, which may overestimate the association of these alterations with this disease.
Future studies should measure SpO 2 , Ct value, GD, and OD changes in a follow-up study at early infection and monitor their levels during several stages of disease progression. Despite evidence of the existence of alteration of taste and smell as a symptom of COVID-19, it is still required to carry out experimental studies that explain the mechanism by which the infection produces this wide range of alterations, as well as their exact prevalence to find effective strategies for prevention, diagnosis, treatment, and rehabilitation of these conditions.

Conclusion
The leading utility of our finding may assist physicians in focusing on measuring SpO 2 , Ct beside D-dimer at the early stage of the disease since both parameters were significantly decreased in COVID-19 patients with a poor prognosis at the time of hospital admission. Besides involving Ct in the mortality of COVID-19, a low value of Ct value is also associated with GD and OD. Age and BMI were also predictors of COVID-19 mortality. Therefore, individuals with low SpO 2 and Ct values but high D-dimer should be monitored carefully to prevent death. The current study's findings suggest that Ct value and its interpretation comments on RT-PCR's report can be used as an early warning system to predict mortality in COVID-19. Once the challenges of Ct value have been overcome, Ct values can be utilized in conjunction with other laboratory biomarkers and clinical characteristics to manage the disease. -607 IL-18 T/G SNP was not associated with mortality in COVID-19. It may be of great diagnostic and therapeutic importance to understand the genetic basis of COVID-19.

Summary points
• It is essential to document the Ct value in the clinical report at the time of admission, since a low Ct value is linked to a higher risk of mortality. • Ct value is also linked to OD and GD.
• -607 IL-18 T/G polymorphism was not associated with mortality in COVID-19 patients. • The regression model suggested that Ct value, age, BMI and D-dimer were predictors of a decrease in SpO 2 .

Supplementary data
To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/ suppl/10.2144/fsoa-2022-0082

Author contributions
All authors contributed equally to this manuscript. SW Smail performed sample collection and data analysis. The serum laboratory biomarkers were measured by E Babaei. All authors discussed the results and contributed to the final manuscript. K Amin wrote the manuscript draft and supervised the conception of this study.